Effects of lithium on receptor-mediated activation of G proteins in rat brain cortical membranes

Neuropharmacology 38 (1999) 403 – 414

Effects of lithium on receptor-mediated activation of G proteins in rat brain cortical membranes Hoau-Yan Wang, Eitan Friedman * Department of Pharmacology, MCP-Hahnemann School of Medicine, 3200 Henry A6enue, Philadelphia, PA 19129, USA Accepted 9 October 1998

Abstract The underlying molecular mechanism of action of lithium in the treatment of manic-depressive illness is not clear. The effect of chronic lithium on GTP-binding and toxin-mediated ADP-ribosylation of specific G proteins in brain cortical membranes was examined. Incubation of cortical membranes with 5-HT increased [35S]GTPgS binding to Gas, Gai, Gao and Gaq proteins. Six weeks but not 1 week of lithium treatment reduced the increases in [35S]GTPgS binding to Gas, Gai and Gao which are produced by 5-HT by 75–85%, whereas 5-HT stimulated [35S]GTPgS binding to Gaq was reduced by 38%. No changes in membrane levels of Ga and Gb proteins were noted in lithium-treated rats. Pertussis toxin (PTX)-mediated ADP-ribosylation of Gai/o was increased by 60% in cortical membranes of chronically treated rats. Lithium treatment did not affect cholera toxin-mediated ribosylation of Gas. Increases in [35S]GTPgS binding to Ga proteins evoked by 5-HT were also inhibited by 0.5 – 2.0 mM lithium chloride added in vitro to the assay mixture. Rubidium and cesium did not change 5-HT-stimulated G protein activation. ADP-ribosylation of Gai/o catalyzed by PTX was not changed by in vitro LiCl. The inhibitions of 5-HT-stimulated increases in [35S]GTPgS-binding to Gas and Gaq were completely suppressed by 2.4 mM MgCl2; this concentration of MgCl2 inhibited the effect of lithium on Gai and Gao by 50%. Similar findings were also noted when [a-32P]GTP was used in the binding assay. The results suggest that lithium interferes with receptor-G protein coupling via a Mg2 + -sensitive mechanism. This action of the drug is more effective for Gs, Gi and Go than for Gq and may result from its interference with the recycling of trimeric G proteins. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: GTP-binding; G proteins; Serotonin receptors

1. Introduction The molecular mechanism by which lithium exerts its clinical action in bipolar affective disorder is not well understood. Considerable amounts of work has recently focused on the effect of lithium on receptorlinked second messenger systems. Lithium was shown to attenuate neurotransmitter-stimulated adenylyl cyclase activity (Ebstein et al., 1980; Newman et al., 1990) and to reduce phosphatidylinositol metabolism (Newman, 1991; Song and Jope, 1992) which may be related to its ability to inhibit inositol-1-phosphatase (Hallcher and Sherman, 1980). Evidence in support of these actions was also obtained in human studies. For exam-

* Corresponding author. Tel.: +1-215-8424203; fax: + 1-2158431515; e-mail: [email protected].  This work was supported by USPHS grant MH45166.

ple, cerebrospinal fluid cyclic AMP concentrations and epinephrine-induced increases in plasma cyclic AMP levels were reduced in patients during treatment with lithium (Friedman et al., 1979) and inositol-1-phosphatase inhibition was shown to occur in human red blood cells during treatment with this ion (Belmaker et al., 1990). The fact that both second messenger systems are regulated by the guanine nucleotide regulatory proteins (G proteins) had led investigators to examine the effects of lithium on G proteins. G proteins convey signals from neurotransmitter receptors to specific intracellular effector systems. In brain, Gas and Gai regulate the activity of adenylyl cyclase and Gao and Gaq couple neurotransmitter receptors to effector systems such as phospholipase C and ion channels (Ohara et al., 1987; Berstein et al., 1992; Jope et al., 1994). The G proteins exist as heterotrimers, which are activated by receptor occupation—

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an event that leads to the exchange of GDP by GTP at the a subunit and its dissociation from the bg dimer. The GTP-bound Ga subunit and the bg complex subsequently interact with intracellular effectors (Tang and Gilman, 1991; Smrcka and Sternweis, 1993). Therapeutic levels of lithium were shown to inhibit both adrenergic and cholinergic agonist-stimulated increases in [3H]GTP binding to rat cerebrocortical membranes (Avissar et al., 1988). These authors have also shown that in manic subjects receptor-activated [3H]5%guanylylimidodiphosphate (Gpp(NH)p) binding to mononuclear leukocyte membranes was reduced by lithium treatment (Schreiber et al., 1991). While these studies implicate the G proteins in the action of lithium a recent study performed in platelets concluded that in vitro lithium does not affect coupling of receptors to Gi protein (Odagaki et al., 1997). Protracted lithium administration was also shown not to affect the levels of Ga proteins (Li et al., 1993; Emamghoreishi et al., 1996). Thus, the mechanism and the specific G protein subtype(s) that may be involved in the actions of lithium have not been defined. In the present communication we report on the effects of lithium treatment on the ability of serotonin receptors to increase GTP binding to specific Ga proteins. 2. Materials and method

2.1. Materials Pertussis toxin (islet-activating protein) was purchased from List Biologicals (Campbell, CA). Leupeptin was purchased from Peptides International (Louisville, KY). Soybean trypsin inhibitor, 5-hydroxytryptamine creatinine sulfate, carbachol chloride, phenylmethylsulfonyl fluoride, Gpp(NH)p, thymidine, dithiothreitol, isonicotinic acid hydrazide, b-nicotinamide adenine dinucleotide (NAD), Cholera toxin and 3-acetylpyridine adenine dinucleotide (3-APAD) from Sigma (St. Louis, MO). [35S]guanosine 5%-O-(3-thiotriphosphate) ([35S]GTPgS, 1311 Ci/mmol), [a 32P]NAD (3000 Ci/mmol) and G protein antipeptide antibodies were purchased from NEN (Boston, MA). [125I]-protein A, [a-32P]GTP (3000 Ci/mmol) was obtained from ICN Biomedical, (Costa Mesa, CA). Normal rabbit serum, recombinant Gas protein and Pansorbin cells were purchased from Calbiochem (La Jolla, CA). Recombinant Gaq protein was purchased from CHEMICON International (Temecula, CA). All other reagents were of analytical grade. Gaz protein antiserum ( c 2919) was kindly provided by Dr David Manning (Department of Pharmacology at the University of Pennsylvania, Philadelphia, PA). Recombinant Gai1 and Gao proteins were kindly provided by Dr Mark M. Rasenick (Department of Physiology and Biophysics at the University of Illinois, Chicago, IL).

2.2. Lithium treatment Male Sprague–Dawley rats (Zivic–Miller, Zelienople, PA) weighing 200–225 g were fed rat chow containing LiCl (0.212%, Teklad Test Diets Madison, WI). Controls were fed similar diet without LiCl for up to 6 weeks. Water and 1.8% saline were provided to all animals ad lib. Animals which received the LiCl diet had serum lithium concentrations of 0.659 0.03 mEq/l.

2.3. Receptor-stimulated GTP binding to specific G-proteins Rats were sacrificed by decapitation and the brains were removed. All procedures were carried out at 4°C unless otherwise indicated. Cortex was dissected and homogenized (glass/glass) in 10 vol. of oxygenated Kreb’s–Ringer buffer (KRB) containing 25 HEPES (pH 7.4), 154 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.2% 2-mercaptoethanol, 50 mg/ml leupeptin, 25 mg/ml pepstatin A, 0.01 U/ml soybean trypsin inhibitor, 0.04 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 750×g for 5 min. The obtained supernatant was further centrifuged at 48 200× g for 10 min and the pellet was resuspended in 10 vol. of KRB and used as the crude membrane preparation. To study the effect of Mg2 + , the tissue was homogenized in Mg2 + -free KRB containing 10 mM ethylenediaminetetraacetic acid, disodium salt (EDTA), the membranes were washed 2 times each with 10 vol. of Mg2 + -free KRB for 5 min followed by centrifugation. Tissues were then resuspended in Mg2 + -free KRB and protein values were determined by the method of Lowry et al. (1951). The assay mixture (250 ml) containing 200 mg of membrane protein, 2 nM [35S]GTPgS (0.6 mCi/assay) was incubated for 5 min at 30°C followed by incubation in the absence or presence of drug for 5 min. To examine the effects of ions, membranes were incubated for 15 min in the presence or absence of the indicated concentrations of LiCl, RbCl or CsCl prior to the addition of [35S]GTPgS. The reaction was terminated by the addition of 250 ml ice-cold KRB containing 2 mM EDTA and centrifugation at 16 000× g for 5 min. In a separate experiment, tissues prepared by homogenizing in 10 volumes of ice-cold homogenization buffer containing 25 mM HEPES (pH 7.4), 100 mM sucrose, 1 mM ethylene glycol-bis(b-aminoethyl ester) N,N,N%,N%-tetraacetic acid (EGTA), 0.2% 2-mercaptoethanol, 50 mg/ml leupeptin, 25 mg/ml pepstatin A, 0.01 U/ml soybean trypsin inhibitor and 0.04 mM phenylmethylsulfonyl fluoride using glass/glass homogenizer. The tissue homogenate was centrifuged for 5 min at 750× g (4°C). The resulted supernatant was then centrifuged for 10 min at 48 200× g (4°C). The pellet was resuspended in KRB and used as the crude

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membrane preparation. Membranes (200 mg) were incubated with KRB containing 5 nM GTP for 2 min followed by an additional incubation with 50 nM of [a-32P]GTP (10 mCi) for 3 min in the absence (control) or presence of various concentrations of 5-HT (total incubation volume, 250 ml). The reaction was terminated by diluting with 750 ml of ice-cold Mg2 + -free, K – R containing 1 mM EGTA, mixed, placed on ice and immediately centrifuged at 16 000× g for 5 min. The obtained pellets were either solubilized in sample preparation buffer (62.5 mM Tris – HCl, pH6,8, 10% glycerol, 2% Sodium dodecyl Sulfate (SDS), 5% 2-mercaptoethanol and 0.1% bromophenol blue) and analyzed using SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography or solubilized followed by immunoprecipitation with specific anti-Ga antisera (1:1000 dilution) using a procedure described previously (Friedman et al., 1993). The resulting pellet containing the antigen-antibody complex was resuspended in KRB by brief sonication and radioactivity was measured by liquid scintillation spectrometry. The radioactivity precipitated by the normal rabbit serum was considered background and subtracted from all agonist stimulated values. In experiments where [a32 P]GTP was used, the [a-32P]guanine nucleotide bound proteins were analyzed by SDS-PAGE (12% polyacrylamide gel) according to Laemmli (1970). The Coomassie blue stained gels were dried and subjected to autoradiography. The autoradiograms were analyzed by densitometric scanning (Zeineh Soft Laser Scanning Densitometer, Fullerton, CA). Previous studies have shown that no cross reactivity was found in the immunoprecipation assay between Gas and Gai proteins, while a 20% crossreactivity was observed between Gai and Gao (Friedman et al., 1993). Furthermore, antisera against Gaz and Gaq did not cross react with any of the Ga proteins examined (data not shown). In a separate set of experiments, the effectiveness of the immunoprecipitation and the effect of lithium on antibody-antigen recognition were determined by the ability of the antisera to precipitate [35S]GTPgS-bound to purified Gas, Gai1 and Gao. In these assays, 50 ng of the respective recombinant Ga protein was incubated in 250 ml of KRB for 30 min with 20 nM [35S]GTPgS at 30°C. A total of 750 ml of the immunoprecipitation buffer was then added and incubation with antisera directed against specific Ga protein was carried out as described above. The amount of [35S]GTPgS in the immunoprecipitates was measured and compared to the total specific bound [35S]GTPgS radioactivity which determined by that retained on the filter when the reaction was terminated by filtering over a 10-kDa molecular weight cut-off filters (Cole – Parmer instrument, Niles, IL). The amount of [35S]GTPgS on the filter was measured by liquid scintillation spectrometry.

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Specific binding was determined by the differences in binding in the presence and absence of 100 mM GTP.

2.4. Immunoblot analysis Membranes from rat brain cortex were prepared as described above. Twenty five micrograms of membranes were solubilized in sample prep. buffer and proteins were separated by SDS-polyacrylamide gel electrophoresis (12% polyacrylamide gel) according to Laemmli (1970). Proteins were then transferred electrophoretically to a nitrocellulose membrane. This amount of protein was within the linear range of detection using saturating dilutions of each antisera. The completeness of transfer was checked by Coomassie blue staining of the gel. The membrane was incubated at room temperature for 2 h in 3% bovine serum albumin in phosphate buffered saline (BSA/PBS) to block nonspecific sites. The membrane was then washed, incubated overnight with antipeptide antisera (diluted 1:1000 in BSA/PBS) and unbound antibody was washed out with PBS containing 0.05% Tween 20 (TBS). Following incubation with [125I]-protein A (500 000 cpm) the blots were extensively washed three times with 0.05% TBS (10 min each), dried and subjected to autoradiography at − 70°C with intensifying screens for 24 h. In some experiments, membranes were incubated at 4°C overnight with 10% non-fat dry milk in 0.1% TBS to block nonspecific sites, washed with 0.1% TBS, incubated for 1 hr with antiserum raised specifically against specific a-subunit of various G protein (1:1000 in 0.1% TBS) and unbound antibody was washed out with 0.1% TBS. Following a 60 min incubation with HRP-conjugated goat anti-rabbit IgG (Amersham) (1:10 000 in 0.1% TBS), the blots were extensively washed with 0.3% TBS. The immunoreactive proteins were detected by the enhanced chemiluminescence (ECL) Western blot detection system and visualized by 10 s exposure to film. The autoradiograms and fluorograms were quantitated by densitometric scanning using Zeineh Soft Laser Scanning Densitometer (Fullerton, CA).

2.5. ADP-Ribosylation with [a 32P]NAD ADP-ribosylation was carried out according to the method of Gill and Woolkalis (1991) with some modifications. Brain cortical tissue was removed, homogenized in 10 vol. of TMES buffer containing 50 mM Tris–HCl PH 7.6, 1 mM EDTA, 2 mM MgCl2, 0.01 U/ml Soybean trypsin inhibitor and 10% sucrose and centrifuged at 1000× g for 5 min. The supernatant was saved and the pellet resuspended in 5 vol. of TMES. After centrifugation at 1000× g for 5 min the supernatant was combined with the first supernatant and centrifuged at 25 000× g for 10 min. The resultant pellet

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Fig. 1. Effects of in vivo and in vitro lithium treatments on 5-HT-stimulated [a-32P]GTP binding to Ga proteins. (A) Cerebrocortical membranes were prepared from control and lithium-treated rats. (E) Cerebrocortical membranes were prepared from control animals and incubated with 1 mM LiCl for 10 min. Tissues (200 mg membrane proteins) were then incubated with [a-32P]GTP (50 nM, 10 mCi) and indicated concentration of Mg2 + followed by stimulation with 10 mM of 5-HT. The [a-32P]guanine nucleotide-labeled Ga proteins were analyzed by 12% SDS-PAGE and autoradiography. The resulting autoradiograms were further evaluated by scanning laser densitometry. Each autoradiograms is a representative of five individual experiments. In (A) and (E) two groups of The [a-32P]guanine nucleotide labeled protein bands were noted with apparent molecular weights of 51- and 40-kDa, respectively. The identities of various protein bands were confirmed by immunoblot analysis with HRP/ECL detection on the same sample from control (C) and 5-HT-activated (S) tissues using specific anti-Ga antisera. ((B) Ga s: 52- and 45-kDa; (C) Gai: 41-kDa; (D) Gao: 40-kDa). Serotonin (5-HT) increased [a-32P]guanine nucleotide binding to Gas and Gai/Gao by around 200 and 180%, respectively. This 5-HT-mediated response was inhibited by both in vivo and in vitro lithium treatments by 90%. Increasing Mg2 + concentration from 1 to 2 mM partially reversed (65% in in-vivo lithium-treated tissues, 42% in in vitro lithium-treated tissues). Arrows mark the apparent molecular weight of 47-kDa. C, control; S, serotonin (10 mM); L, lithium-treated; LS, lithium-treated with 10 mM 5-HT stimulation.

was resuspended in HNT buffer (10 mM HEPES, pH 7.3, 130 mM NaCl, 0.01 U/ml Soybean trypsin inhibitor and 0.2% triton X-100) and 1 ml aliquots were frozen and stored at − 70°C. At the time of the assay, an aliquot was thawed and protein concentration was determined using the method of Lowry et al. (1951). Fifty micrograms of membrane protein was incubated with (cholera toxin: CTX) or without (pertussis toxin, islet activating protein: PTX) 1 mM Gpp(NH)p in a total volume of 10 ml at 25°C for 10 min. Ten microlitres of [32P]NAD mixture and 2 ml of CTX or PTX [final concentration in each assay in total of 22 ml containing 10 mM HEPES pH 7.3, 130 mM NaCl, 0.01 U/ml Soybean trypsin inhibitor, 10 mM thymidine, 10 mM dithiothreitol, 20 mM isonicotinic acid hydrazide, 1 mM 3-APAD, 20 mM [32P] NAD (10 000 cpm/pmol), 200 mg/ml activated toxin, 0.1% Triton X-100 and 1 mM ATP (for pertussis toxin treatment only), 0.5 mM Gpp(NH)p (for cholera toxin treatment only)]. Incubation was carried out at 25°C for 60 min. The reaction was stopped by the addition of 0.1 ml of ice cold HEPES buffer pH 7.3 containing 130 mM NaCl (HN buffer) followed by centrifuging at 16 000×g for 20 min. For CTX-treated samples, the membrane pellet was washed once and resuspended in HN buffer and sample preparation buffer was added. To separate Gai from Gao in PTX-treated samples, the pellets were resuspended and heated at 75°C for 5 min in 15 ml of buffer containing 40 mM Tris – HCl (pH 6.8), 1 mM

dithiothreitol (DTT) and 2% SDS. Ten microlitres of 100 mM N-ethylmaleimide (NEM) was then added and reaction was performed at 25°C for 15 min followed by 2 min boiling. The samples were subjected to SDSPAGE. The Coomassie blue stained gels were dried and subjected to autoradiography. ADP-ribosylation was quantitated by densitometric scanning of the autoradiograms.

2.6. Statistical analysis Statistical differences between groups were analyzed by two-tailed Student’s t-tests. Dose-response curves were evaluated by two-factor analysis of variance (ANOVA) and comparisons among the means were made by Newman–Keuls or Dunnett’s tests. The threshold for significance was PB0.05.

3. Results

3.1. Serotonin-induced GTP binding to cerebrocortical membrane Ga proteins Incubation of cortical membranes with 5-HT in the presence of [a-32P]GTP was found to increase [32P]guanine nucleotide binding to two groups of proteins with apparent molecular weights of 51- and 40-kDa (Fig. 1A and E). The [a-32P] guanine nucleotide

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Fig. 2. Lithium-treatment diminished the 5-HT-stimulated [a-32P]GTP binding to Gas, Gai, Gao and Gaq proteins. A representative autoradiogram of lithium’s influence on the effect of 10 mM 5-HT-stimulated [a-32P]GTP binding to Gas, Gai, Gao (A) and Gaq (B) proteins without affecting [a-32P]GTP binding to Gaz is shown. Cerebrocortical membranes (200 mg) from control and lithium-treated rats were incubated with 5 nM GTP for 2 min followed by incubation with 50 nM [a-32P]GTP (10 mCi) and 10 mM 5-HT for 3 min. Reactions were terminated by dilution and centrifugation. The [a-32P]guanine nucleotide-bound Ga proteins were immunoprecipitated with anti-Ga sera and separated by SDS-PAGE. The representative autoradiogram of 4 individual experiments indicates that lithium treatment abolished the 5-HT-induced increase in [a-32P] guanine nucleotide binding to Gas, Gai and Gao while partially inhibited 5-HT responsive [a-32P] guanine nucleotide binding to Gaq. In contrast, binding to Gaz was neither enhanced by inclusion of 5-HT nor affected by lithium treatment. C, control; S, 5-HT (10 mM); L, lithium-treated; LS, lithium-treated with 10 mM 5-HT stimulation.

labeled proteins comigrated with Gas (51-kDa) and Gai, Gao, Gaz and Gaq (40-kDa) which were identified by immunoblots obtained from the same gels that were used in the autoradiography (Fig. 1B, C and D). These immunoblots also show that the levels of membrane Ga proteins (Gas, Gai, Gao) are not changed by incubation with 5-HT (Fig. 1B, C and D). Similarly, Gaz and Gaq which co-migrated with Gai and Gao proteins were also not affected by the incubation of the membranes with 5-HT (data not shown). When individual Ga proteins were immunoprecipitated from solubilized membranes by anti-Ga antisera and separated on SDS-PAGE, single [32P]-labeled bands were noted on each autoradiogram. These bands corresponded to Gas (48-kDa), Gai (41-kDa), Gao (40-kDa), Gaz (42-Da) and Gaq (41-kDa) proteins. Incubation of membranes with 10 mM 5-HT increased [32P]GTP binding to the Ga proteins in the following order: Gao ]Gai ]Gaq\ Gas (Fig. 2). Serotonin did not increase [32P]GTP

binding to Gaz (Fig. 2). Activation of membrane serotonin receptors was also found to increase binding to Ga proteins when the nonhydrolyzable GTP analog, [35S]GTPgS was used. The neurotransmitter increased [35S]GTPgS binding to Gas, Gai, Gao and Gaq/11 in a concentration-related fashion (Fig. 3). At the maximal concentration of 10 mM, serotonin induced 2.5-, 4.5-, 5.5- and 3.5-fold increases in [35S]GTPgS binding to Gas, Gai, Gao and Gaq, respectively (Figs. 3 and 4). Serotonin did not increase [35S]GTPgS binding to Gaz (Fig. 3). The effectiveness of the immunoprecipitation of Ga proteins and the magnitude of the serotonin-induced activation of the G proteins does not appear to be a function of the differences in cortical membrane Ga protein levels. This is implied by the demonstration that although the respective levels of Gas, Gai, Gao and Gaq proteins in cortical membranes were 0.08, 0.15, 0.75 and 0.12% of total membrane protein (Table 1), 80–86% of the Ga proteins was precipitated by the

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respective antisera using Western blot analyses. Furthermore, the ability of anti-Ga antisera to precipitate recombinant Ga proteins was also examined by comparing the amount of [35S]GTPgS bound to immunoprecipitated recombinant Ga protein as a fraction of the total protein bound [35S]GTPgS which is retained on a 10-kDa molecular weight cut-off filters. A calculated 90% recovery of the total protein was obtained.

3.2. The effect of lithium on 5 -HT-induced GTP binding to specific Ga proteins in cortical membranes The effect of chronic lithium administration on 5-HT activated [a-32P]GTP binding to Ga proteins was examined. Fig. 1A and E demonstrate that 5-HT stimulated binding of [a-32P]GTP to the 51- and 40-kDa protein bands were inhibited in cortical tissues from chronically lithium-treated rats. Immunoprecipitating the specific Ga proteins from solubilized membranes revealed that chronic lithium administration markedly inhibited 5HT-stimulated [a-32P]GTP binding to Gas, Gai, Gao and Gaq proteins (Fig. 4). It is also apparent that the effect of the treatment was greater for Gas, Gai and Gao than for Gaq protein. Serotonin-induced increases in [35S]GTPgS binding to specific Ga proteins were also examined in membranes obtained from control and lithium-treated rats. Fig. 4 demonstrates that the 5-HTmediated increases in [35S]GTPgS binding to Gs, Gi or Go protein were reduced by 75 – 85% in animals treated

Fig. 3. Serotonin-induced [35S]GTPgS binding to Ga proteins. Cerebral cortical membranes prepared from rats were incubated with 2 nM [35S]GTPgS and stimulated with 1 nM–100 mM of 5-HT. Immunoprecipitated [35S] labeled Gas, Gai Gao, Gaz and Gaq were assessed. Basal [35S]GTPgS binding to Gas, Gai, Gao, Gaz and Gaq (in CPM) were 975.69 85.7, 1014.39 108.6, 1103.59 112.7, 764.2 9 59.8 and 995.4 981.8, respectively. The data expressed as increased [35S]GTPgS binding in the presence of 5-HT as compared to basal [35S]GTPgS binding is depicted as the mean 9SEM of five experiments. Statistical analyses using ANOVA reveals that 5-HT increased [35S]GTPgS binding to Gas, Gai, Gao and Gaq in a concentrationdependent manner (P B0.01).

Fig. 4. Effect of 6- and 1-week lithium treatment on 5-HT-induced [35S]GTPgS binding to Ga proteins. Cerebral cortical membranes were prepared from control (C) and lithium-treated (L) rats and incubated with 10 mM of 5-HT in the presence of 2 nM [35S]GTPgS. Immunoprecipitated [35S] labeled Gas, Gai Gao, Gaz and Gaq were assessed. The data expressed as increased [35S]GTPgS binding in the presence of agonist as compared to [35S]GTPgS binding in the absence of agonist (basal) is depicted as the mean 9SEM obtained from five to six separate treatment groups. In control brains, basal [35S]GTPgS binding to Gas, Gai, Gao, Gaz and Gaq (in CPM) were 1209.5 9103.5, 1265.19 38.2, 1331.19 72.0, 917.2973.5 and 1281.4 956.8, respectively. In 1-week lithium-treated brains, basal [35S]GTPgS binding to Gas, Gai, Gao, Gaz and Gaq (in CPM) were 1029.3 989.4, 1185.2987.6, 1219.59 97.4, 805.39 73.5 and 1132.1 991.3, respectively. In 6-week lithium-treated brains, basal [35S]GTPgS binding to Gas, Gai, Gao, Gaz and Gaq (in CPM) were 1175.4 956.6, 1277.7938.5, 1350.99 100.9, 898.5967.2 and 1207.6 9101.3, respectively. The effect of 6-week but not 1-week lithium treatment was significantly different from control for respective Ga proteins and agonists (* PB 0.01, Dunnett’s tests).

for 6 weeks with the lithium-containing diet. Serotonin stimulated [35S]GTPgS binding to Gaq was, however, inhibited by only about 38% by the drug treatment. Basal [35S]GTPgS binding to the Ga proteins was not altered by lithium treatment. A 1-week treatment regimen with the LiCl-containing diet did not influence [35S]GTPgS binding to the Ga proteins. In in vitro experiments, 5-HT-induced increases in [a-32P]GTP or [35S]GTPgS binding to cerebrocortical membrane Ga proteins were found to be inhibited by 0.5–2.0 mM LiCl. However, basal binding was unchanged by these concentrations of LiCl (Fig. 1E and Fig. 5). In contrast to the effects of LiCl, addition of 2 mM RbCl or CsCl did not influence receptor-stimulated [35S]GTPgS binding (Fig. 5). In a separate set of experiments, the effect of in vitro LiCl on [35S]GTPgS binding to recombinant Ga proteins was determined. The results indicate that binding of [35S]GTPgS to Gas, Gai1 or Gao were not affected by the presence of 1 mM LiCl (data not shown). The results therefore, indicate that lithium, both added in vitro to brain membranes or during chronic in vivo treatment impairs 5-HT-stimulated GTP binding to membrane Ga proteins but does not

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Table 1 Quantitation of membrane Ga proteins in rat cortexa Density in arbitrary units Standard (50 ng)

25 mg membrane proteins

Absolute quantity (ng)

%

Gas Gai Gao Gaq

4404.09 389.5 8571.19 1325.7 44917.39 3819.5 6044.19 843.5

20.8 38.3 185.2 29.0

0.08 0.15 0.75 0.12

10586.59 1432.9 11189.4 9 2123.5 12126.79 1957.8 10421.49 1013.2

a The levels of Ga proteins in rat cortical membranes were evaluated by immunoblots using anti-peptide antisera raised against specific Ga proteins. Twenty-five mg of solubilized rat cerebrocortical membranes and 50 ng of purified recombinant Ga proteins were separated on 10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes and immunoblotted with anti-Ga antisera with HRP/ECL detection. Densitometric analysis of the immunoblots is expressed in arbitrary units. The absolute levels of expression for each individual Ga proteins are calculated by comparing with the density of the respective recombinant Ga protein. The percentage of each Ga protein as a fraction of total in membrane protein is obtained by comparing the calculated absolute expression level to the total membrane protein.

change basal binding of GTP to Ga proteins under the tested conditions. The inhibition of G protein activation observed in tissue obtained from chronic lithium treated animals is not caused by a reduction in Ga or Gb proteins since treatment with lithium for 6 weeks did not change the levels of membrane Gas, Gai, Gao, Gaz or Gaq; nor was the amount of membrane-associated Gb protein altered by chronic lithium treatment (Fig. 6). In cortical homogenates of both control and lithium-treated animals, the Ga proteins were almost exclusively associated with cortical membranes since only negligible amounts of Ga protein immunoreactiv-

ities were detected in the cytosolic fractions of the homogenates (data not shown). The effect of chronic lithium treatment on the effectiveness of the immunoprecipitation procedure, assessed by immunoblots, indicated that exposure to the ion did not affect the recovery of the specific Ga proteins precipitated by the respective antisera (data not shown). Similarly, lithium did not affect antibody-antigen interaction as comparable levels of [35S]GTPgS bound to recombinant Ga proteins were immunoprecipitated with antiGa antisera when the reaction was performed in the presence or absence of 1 mM LiCl.

3.3. Effects of lithium on ADP-ribosylation of Ga proteins

Fig. 5. Effect of in vitro lithium, rubidium and cesium on 5-HT-induced [35S]GTPgS labeling of Ga proteins. Cerebrocortical membranes were prepared from control rats and responses to 10 mM 5-HT were tested in the presence of LiCl, RbCl or CsCl at the indicated concentrations. Binding of [35S]GTPgS to specific immunoprecipitated Ga proteins was measured. The data are expressed as increased [35S]GTPgS binding in the presence of agonist as compared to [35S]GTPgS binding in the absence of agonist. Basal [35S]GTPgS binding to Gas, Gai, Gao, Gaz and Gaq (in CPM) in control (vehicle-treated) membranes were 1015.2 9 97.2, 1189.39 91.4, 1346.79 102.8, 879.5 9 58.9 and 1156.79 86.2, respectively. The data are the mean 9 SEM obtained from five preparations. The statistical significance was determined by the two-factor ANOVA followed by the Dunnett’s test. * PB 0.05 for all four 5-HT responsive Ga proteins when compared to control (C) binding.

The effects of lithium on CTX- and PTX- mediated ADP-ribosylation of Ga proteins was examined in cerebrocortical membranes taken from rats which were treated for 6 weeks with lithium diet. In preliminary experiments, concentrations of CTX or PTX which elicited optimal ribosylations in control cortical tissues were selected. Fig. 7 demonstrates that CTXcatalyzed ADP-ribosylation of membrane Gas was not altered by lithium treatment when compared to tissue from control animals. In contrast, PTX-mediated ADP-ribosylation of Gai/o was enhanced by about 60% in cortical membranes taken from lithiumtreated animals (Fig. 8). This lithium-mediated effect was not accompanied by an alteration in Gai/Gao levels as measured by Western blots (data not shown). The addition of up to 4 mM LiCl to control membranes, either immediately or 30 min prior to the addition of [a 32P]NAD did not change PTX-catalyzed ADP-ribosylation. Similarly, treatment with lithium for 1 week did not alter PTX-mediated ADP-ribosylation of Gi/o (data not shown). The results suggest that long-term (6 weeks) but not short term (1 week) or in vitro treatment increases the ADP-ribosylation of membrane Gi and Go proteins.

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Fig. 6. Immunoblot analysis of Ga and Gb proteins. The levels of Ga proteins in rat cortical membranes were evaluated by immunoblots using anti-peptide antisera raised against specific Ga proteins. Representative autoradiograms of the immunoblots for the indicated Ga and Gb proteins using [125I]-Protein A for detection are shown ((A), Gas, Gai, Gao and Gb; (B) Gaz and Gaq). Densitometric analysis of the immunoblots is depicted in the bar graph expressed as area of density in arbitrary units (C). The empty bars represent the data from the control group and the solid bars represent the data from lithium-treated group. There were no significant differences between control and lithium treatment for any of the Ga and Gb proteins. Data are mean 9 SEM of membranes from four separate experimental groups.

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the concentration-response curve of Mg2 + to right, however, at 1.2 mM Mg2 + - the concentration used in the assay-lithium did not affect the magnitude of [35S]GTPgS bound to Gao (data not shown).

4. Discussion

Fig. 7. Effect of chronic lithium treatment on CTX activated ADP-ribosylation of cerebrocortical membrane G proteins. Representative autoradiograms of Gas ribosylated in the presence of [32P]NAD, Gpp(NH)p and activated by CTX is shown in control and lithiumtreated tissues from two individual animals (Top). The CTX sensitive 32 P-labeled bands that comigrate with the two forms of Gas (51- and 45-kDa) were analyzed by densitometric scanning and are expressed as an area of density in arbitrary units (Bottom). The results shown in the bar graph are expressed as the mean9 SEM obtained from four independent treatment groups (empty bars represent the data from control tissues, solid bars represent the data from lithiumtreated tissues). No significant difference between control and lithium treatment was observed (two-tailed Student’s t-test).

3.4. The effect of Mg 2 + on the inhibitory action of lithium on 5 -HT-induced GTP binding The data summarized in Fig. 9 demonstrate that 5-HT stimulated [35S]GTPgS binding to Ga proteins is Mg2 + -dependent. The effects of lithium on 5-HT-induced increases in [35S]GTPgS binding to Gas and Gaq were completely blocked by 2.4 mM MgCl2. However, the effects of lithium on Gai and Gao were only partially inhibited by the same concentration of MgCl2 (Fig. 9). In agreement, 2 mM Mg2 + also appeared to partially reverse the effects of lithium on 5-HT-induced elevations in [a-32P]GTP binding to Ga proteins (Fig. 1A and E). The effect of LiCl on the Mg2 + -dependent binding of [35S]GTPgS to recombinant Gao was also assessed. The results indicate that 1 mM LiCl shifted

The present data demonstrate that lithium interferes with signal transduction of cortical serotonin receptors. Serotonin-mediated increases in binding of [32P]GTP or of [35S]GTPgS to rat brain cortical membrane Ga proteins were attenuated by a long-term (6 weeks) lithium treatment regimen which results in therapeutic plasma ion concentrations. Lithium ion was also found to block the increase in receptor-mediated GTP binding to the Ga proteins when added in vitro to a membrane preparation at concentrations of 0.5–2.0 mM but not after 7 days of treatment with a lithium-containing diet, suggesting that a protracted treatment schedule may be required for sufficient accumulations of the drug in brain tissue or in a particular intracellular compartment. The specificity of lithium in preventing receptoractivated GTP binding to specific Ga proteins is demonstrated by the ineffectiveness of Rb + or Cs + salts to alter 5-HT-induced increases in GTP binding. The present data supplement a previous report which noted that lithium, both in vitro and after 21 days of in vivo treatment, inhibited [3H]GTP binding to cerebrocortical membranes in response to b-adrenergic or cholinergic receptor stimulation (Avissar et al., 1988). The inhibition of receptor-mediated GTP binding to Ga proteins by lithium is clearly not due to a change in the steady-state concentrations of Ga proteins in cortical membranes of treated animals as reported here and previously presented by other workers. The findings that basal [35S]GTPgS binding was unchanged by the in vivo lithium treatment also support this conclusion (Li et al., 1993). Furthermore, the similarity between the magnitudes of the effects of lithium when employing [a-32P]GTP or [35S]GTPgS in the binding assay suggests that the effect of lithium is not mediated by alteration in GTPase activity intrinsic to the Ga -subunits since the thio derivative of GTP is a poor substrate for GTPase. Thus, the data suggest that lithium ion interferes with receptor-stimulated G protein activation, but not with binding of the guanine nucleotide to Ga proteins under non-stimulated conditions. The present and previous data (Avissar et al., 1988; Schreiber et al., 1991; Carli et al., 1994) indicate that lithium interferes with the flow of signals from receptors to their associated G protein(s). Since this effect of lithium was observed for diverse G protein-coupled receptor systems and for different G proteins, it is hypothesized that the ion interferes with receptor stimulated receptor/G protein coupling. Functional uncou-

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Fig. 8. Effect of chronic lithium treatment on PTX activated ADP-ribosylation of cerebrocortical membrane G proteins. Representative autoradiograms of Gai/Gao ribosylated in the presence of [32P]NAD and activated by PTX is shown in duplicate for control and lithium-treated tissues (Top). The PTX sensitive 32P-labeled protein band was analyzed by densitometric scanning and are expressed as an area of density in arbitrary units (Bottom). The results shown in the bar graph are expressed as the mean9SEM obtained from four independent treatment groups (empty bars represent the data from control tissues, solid bars represent the data from lithium-treated tissues). Significance between control and lithium treatment was observed (* PB 0.01; two-tailed Student’s t-test).

pling of membrane receptors from G proteins may result from lithium-elicited changes at the level of receptor, G protein or other cell membrane proteins that regulate signaling via G protein-coupled receptors. The possibility that lithium elicited alterations in membrane phospholipid constituents (Miller et al., 1990) and/or in membrane fluidity (Lopez-Corcuera et al., 1988) contributes to the apparent altered receptor/G protein coupling can not be excluded. Furthermore, lithium does not uniformly alter the binding parameters of the serotonin receptors, suggesting that this effect of the drug is unlikely mediated by changes in receptor number or affinity. Nonetheless, lithium may induce post-translational modifications of G-protein coupled receptors that result in changes in their ability to interact with G proteins. While chronic lithium treatment did not alter the concentrations of the Ga proteins as demonstrated by the immunoblot analyses, changes at the trimeric G protein level are suggested by the marked increase in PTX-activated ADP-ribosylation of membrane Gai/ Gao proteins since it is the trimeric state of these G proteins that is the substrate for PTX ribosylation (Bokoch et al., 1984). While increased ribosylation was found after chronic lithium treatment, no changes in Gai/o ADP-ribosylation were observed under in vitro conditions that also reduce receptor-mediated activation of the Ga proteins. This difference suggests that lithium interferes directly with receptor-mediated G protein activation and after long-term treatment, leads to the accumulation of trimeric (undissociated) G proteins. It appears that a sustained blockade of G protein activation during long-term treatment with the ion is required before increases in trimeric Gi/o are achieved. Increases in PTX-catalyzed ADP-ribosylation of Gai/o were previously reported in rat brain cortex after chronic lithium treatment (Mosana et al., 1992), in

platelets and leukocytes obtained from bipolar affective disorder patients during treatment with lithium (Hsiao et al., 1992; Manji et al., 1995) or following termination of long-term lithium treatment (Watanabe et al., 1990). However, others have reported that chronic lithium treatment decreases (Watanabe et al., 1991) or does not change (Odagaki et al., 1991) PTX-catalyzed ADP-ribosylation of Gai/o. These apparent discrepancies may result from differences in subunit composition that make up the specific Gi and Go proteins or in ribosylation factors found in the various tissues (Kawamoto et al., 1991). Alternatively, differences in length of drug treatment, in methods used in preparing membranes or in other experimental conditions and tissues employed in the various studies may contribute to the discrepancies in results. Nonetheless, functional uncoupling of receptor from G protein during lithium treatment is supported by studies that demonstrated an elevation in ratio of low/high affinity state of b-adrenoceptors in human lymphocytes (Risby et al., 1991), an increase in inhibitory constant for the high affinity D1 and D2 dopamine receptor binding sites (Carli et al., 1994), an inhibition in GTP-induced shift in agonist affinity state of a2-adrenoceptors in platelets (Watanabe et al., 1991) and a physical dissociation of serotonin receptors from their G proteins (Friedman and Wang, 1995). This lithium-induced reduction in coupling of receptor to its associated G protein subsequently results in a less efficient receptor-mediated effector activation and much higher agonist concentrations may be required to achieve functional results in lithium-exposed tissues (Newman et al., 1990; Song and Jope, 1992). The finding that increasing Mg2 + concentrations in the assay mixture can overcome the inhibitory action of lithium on receptor-stimulated GTP binding to Ga proteins suggests that both the in vivo and in vitro

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actions of lithium are mediated by competing with Mg2 + . However, at the 1 mM Mg2 + concentration routinely used in this investigation, lithium did not affect basal GTP binding to either membrane or recombinant Ga proteins. This apparent selectivity in the action of Mg2 + and the previously discussed observation that lithium does not influence basal GTP binding suggest that lithium competes with Mg2 + only at sites

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which are involved in receptor-mediated activation of the G proteins or in their effective coupling to receptors but not at the Mg2 + - regulatory site that modulates GTP binding to Ga subunits. Magnesium ions are known to have multiple sites of action on G proteins (Herrmann et al., 1989; Higashijima et al., 1990). They are essential in the binding of GTP to the a subunit as well as in facilitating effective coupling of receptors to trimeric G proteins (Birnbaumer, 1990; Shiozaki and Haga, 1992). The present data is consistent with a previous report which demonstrated that magnesium diminished the inhibitory actions of lithium on isoproterenol and carbachol-evoked GTP-binding to cerebral cortical membranes (Avissar et al., 1991). The ability of magnesium to antagonize the action of lithium is also consistent with its inhibitory effects on lithium-induced suppression of norepinephrine- or isoproterenol-stimulated adenylyl cyclase (Lonati-Galligani et al., 1989; Mork, 1990) and a2-adrenoceptor mediated inhibition of adenylyl cyclase (Watanabe et al., 1991). The data presented suggest that lithium uncouples serotonin receptors from their associated G proteins. The results furthermore indicate that lithium may exert this action by interfering with Mg2 + at a site that is activated by receptor occupation. In addition, chronic drug treatment was found to elicit an accumulation of undissociated heterotrimeric PTX-sensitive G proteins, a finding which suggest that chronic lithium may increase the stability of trimeric G proteins. The ability of lithium to inhibit receptor-stimulated GTP binding to G proteins may be the relevant action of the drug that mediates its clinical effectiveness in the treatment bipolar affective disorder. This is particularly attractive since enhanced coupling of receptors to G proteins was found in postmortem brains of bipolar affective disorder subjects (Friedman and Wang, 1996). References

Fig. 9. Magnesium reversal of lithium inhibition of 5-HT-induced [35S]GTPgS binding to Ga proteins. Cerebral cortical membranes from control and 6 week lithium-treated rats were prepared in 10 mM EDTA-containing KRB without MgCl2. The tissues were then washed with 10 vol. of Mg2 + -free KRB twice, centrifuged and resuspended in Mg2 + -free KRB. The indicated concentrations of MgCl2 were added to the assay mixtures and 5-HT (10 mM) induced increases in [35S]GTPgS binding to immunoprecipitated Ga proteins were measured. The data are the mean 9SEM from four to five individual experiments expressed as percent of the maximal 5-HTevoked responses in the presence of 1.2 mM MgCl2. The solid symbols represent the control group and the clear symbols represent the lithium treated group. The statistical significance was determined by the two-factor ANOVA followed by the Newman–Keuls test. * P B 0.05 for 5-HT response in lithium-treated group when compared to respective control response.

Avissar, S., Schreiber, G., Danon, A., Belmaker, R.H., 1988. Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex. Nature (Lond.) 31, 440 – 442. Avissar, S., Murphy, D.L., Schreiber, G., 1991. Magnesium reversal of lithium inhibition of b-adrenergic and muscarinic receptor coupling to G proteins. Biochem. Pharmacol. 41, 171 – 175. Belmaker, R.H., Livne, A., Agam, G., Moscovich, D.G., Grisaru, N., Schreiber, G., Avissar, S., Danon, A., Kofman, O., 1990. Role of inositol-1-phosphatase inhibition in the mechanism of action of lithium. Pharmacol. Toxicol. 66 (Suppl. 3), 76 – 83. Berstein, G., Blank, J.L., Smrcka, A.V., Higashijima, T., Sternweis, P.C., Exton, J.H., Ross, E.M., 1992. Reconstitution of agoniststimulated phosphatidylinositol-4, 5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq,11 and phospholipase C-b1. J. Biol. Chem. 267, 8081 – 8088. Bokoch, G.M., Katada, T., Northup, J.K., Ui, M., Gilman, A.G., 1984. Purification and properties of the inhibitory guanine nucleotide binding regulatory component of adenylate cyclase. J. Biol. Chem. 259, 3560 – 3567.

414

H.-Y. Wang, E. Friedman / Neuropharmacology 38 (1999) 403–414

Birnbaumer, G. 1990. Proteins in signal transduction. Ann. Rev. Pharmacol. Toxicol. 30, 675–705. Carli, M., Anand- Srivastava, M.B., Molina-Holgado, E., Dewar, K.M., Reader, T.A., 1994. Effects of chronic lithium treatments on central dopaminergic receptor systems: G proteins as possible targets. Neurochem. Int. 24, 13–22. Ebstein, R.P., Hermoni, M., Belmaker, R.H., 1980. The effect of lithium on noradrenaline-induced cyclic AMP accumulation in rat brain: inhibition after chronic treatment and absence of supersensitivity. J. Pharmacol. Exp. Ther. 213, 161–167. Emamghoreishi, M., Warsh, J.J., Sibony, D., Li, P.P., 1996. Lack of effect of chronic antidepressant treatment on Gs and Gi a-subunit protein and mRNA levels in the rat cerebral cortex. Neuropsychopharmacology 15, 281–287. Friedman, E., Oleshansky, M.A., Moy, P., Gershon, S., 1979. Lithium and catecholamine-induced plasma cyclic AMP elevation. In: Cooper, T.B., Gershon, S., Kline, N.S., Schou, M. (Eds.), Lithium Exerpta Medica. Elsevier, North Holland, pp. 730 – 735. Friedman, E., Butkerait, P., Wang, H.Y., 1993. Analysis of receptorstimulated and basal guanine nucleotide binding to membrane G proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 241, 171–178. Friedman, E., Wang, H.Y., 1995. Lithium interferes with coupling of serotonin receptors with G proteins. ICNP abstract. Friedman, E., Wang, H.Y., 1996. Receptor-mediated activation of G proteins is increased in postmortem brains of bipolar affective disorder subjects. J. Neurochem. 67, 1145–1152. Gill, D.M., Woolkalis, M.J., 1991. Cholera toxin-catalyzed [32P]ADPribosylation of proteins. Meth. Enzymol. 195, 267–280. Hallcher, L.M., Sherman, W.R., 1980. The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatase from bovine brain. J. Biol. Chem. 255, 10896–10901. Herrmann, E., Gierschik, P., Jakobs, K.H., 1989. Neomycin induces high-affinity agonist binding of G-protein-coupled receptors. Eur. J. Biochem. 185, 677 –683. Higashijima, T., Burnier, J., Ross, E.M., 1990. Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J. Biol. Chem. 265, 14176–14186. Hsiao, J.K., Manji, H.K., Chen, G.A., Bitran, J.A., Risby, E.D., Potter, W.Z., 1992. Lithium administration modulates platelet Gi in humans. Life Sci. 50, 227–233. Jope, R.S., Song, L., Powers, R., 1994. [3H]PtdIns hydrolysis in postmortem human brain membranes is mediated by the Gproteins (Gq/11) and phospholipase C-b. Biochem. J. 304, 655 – 659. Kawamoto, H., Watanabe, Y., Imaizumi, T., Iwasaki, T., Yoshida, H., 1991. Effects of lithium ion on ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin, islet-activating protein. Eur. J. Pharmacol. 206, 33–37. Laemmli, U.K., 1970. Cleavage of structural protein during assembly of the head of bacteriophageT4. Nature (Lond.) 227, 680 – 685. Li, P.P., Young, L.T., Tam, Y.K., Sibony, D., Warsh, J.J., 1993. Effect of chronic lithium and carbamazepine treatment on Gprotein subunit expression in rat cerebral cortex. Biol. Psychiatry 34, 162 – 170. Lopez-Corcuera, B., Gimenez, C., Aragon, C., 1988. Changes of synaptic membrane lipid composition and fluidity by chronic administration of lithium. Biochem. Biophys. Acta 939, 467 – 475. Lonati-Galligani, M., Emrich, H.M., Raptis, C., Pirke, K.M., 1989. Effect of in vivo lithium treatment on ( − )isoproterenol-stimulated cAMP accumulation in lymphocytes of healthy subjects and

.

patients with affective psychoses. Pharmacolpsychiatry 22, 241– 245. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265 – 275. Miller, B.L., Lin, K.M., Djenderedjian, A., Tang, C., Hill, E., Fu, P., Nuccio, C., Jenden, D.J., 1990. Changes in red blood cell choline and choline-bound lipids with oral lithium. Experientia 46, 454– 456. Manji, H.K., Chen, G., Shimon, G.H., Hsiao, J.K., Potter, W.Z., Belmaker, R.H., 1995. Guanine nucleotide-binding proteins in bipolar affective disorder. Effect of long-term lithium treatment. Arch. Gen. Psychiatry 52, 135 – 144. Mork, A., 1990. Actions of lithium on second messenger activity in the brain: the adenylate cyclase and phosphoinositide systems. Lithium 1, 131 – 147. Mosana, M.I., Bitran, J.A., Hsiao, J.K., Potter, W.Z., 1992. In vivo evidence that lithium inactivates Gi modulation of adenylate cyclase in brain. J. Neurochem. 59, 200 – 205. Newman, M.E., Drummer, D., Lerer, B., 1990. Single and combined effects of desipramine and lithium on serotonergic receptor number and second messenger function in rat brain. J. Pharmacol. Exp. Ther. 252, 826 – 831. Newman, M.E., 1991. Lithium and the phosphoinositide hydrolysis second messenger system: a critical review of its therapeutic relevance. Lithium 2, 187 – 194. Odagaki, Y., Koyama, T., Yamashita, I., 1991. Lithium does not alter ADP-ribosylation of Gi/Go catalyzed by pertussis toxin in rat brain. Pharmacol. Toxicol. 69, 355 – 360. Odagaki, Y., Nishi, N., Koyama, T., 1997. Lack of interfering effects of lithium on receptor/G protein coupling in human platelet and rat brain membranes. Biol. Psychiatry 42, 697 – 703. Ohara, K., Haga, K., Bernstein, G., Haga, T., Ichiyama, A., Ohara, K., 1987. The interaction between D-2 dopamine receptors and GTP binding proteins. Mol. Pharmacol. 33, 290 – 296. Risby, E.D., Hsiao, J.K., Manji, H.K., Bitran, J., Moses, F., Zhou, D.F., Potter, W.Z., 1991. The mechanisms of action of lithium: II. effects on adenylate cyclase activity and b-adrenergic receptor binding in normal subjects. Arch. Gen. Psychiatry 48, 513–524. Schreiber, G., Avissar, S., Danon, A., Belmaker, R.H., 1991. Hyperfunctional G-proteins in mononuclear leukocytes of patients with mania. Biol. Psychiatry 29, 273 – 280. Shiozaki, K., Haga, T., 1992. Effects of magnesium ion on the interaction of atrial muscarinic acetylcholine receptor and GTP binding regulatory proteins. Biochemistry 31, 10634 – 10642. Smrcka, A.V., Sternweis, P.C., 1993. Regulation of purified subtypes of phosphatidylinositol-specific phospholipase Cb by G protein a and bg subunits. J. Biol. Chem. 268, 9667 – 9674. Song, L., Jope, R.S., 1992. Chronic lithium treatment impairs phosphatidylinositol hydrolysis in membranes from rat brain regions. J. Neurochem. 58, 2200 – 2206. Tang, W.J., Gilman, A.G., 1991. Type-specific regulation of adenylyl cyclase by G protein bg subunits. Science 254, 1500 – 1503. Watanabe, Y., Morita, H., Imaizumi, T., Takeda, M., Hariguchi, S., Nishimura, T., Yoshida, H., 1990. Changes of ADP-ribosylation of GTP-binding protein by pertussis toxin in human platelets during long-term treatment of manic depression with lithium carbonate. Clin. Exp. Pharmacol. Physiol. 17, 809 – 812. Watanabe, Y., Kawamoto, H., Imaizumi, T., Sakagoshi, N., Iwakura, K., Morita, H., Iwasaki, K., Yoshida, H., 1991. Effects of lithium ion on the inhibitory GTP-binding protein and its coupling response. Cell Signal 3, 59 – 64.